DNA Extraction PDF

# DNA Extraction DNA isolation is a process of purification of DNA from sample using a combination of physical and chemical methods. The first isolation of DNA was done in 1869 by Friedrich Miescher. Currently it is a routine procedure in molecular biology or forensic analyses. ## Basic procedur...

# DNA Extraction DNA isolation is a process of purification of DNA from sample using a combination of physical and chemical methods. The first isolation of DNA was done in 1869 by Friedrich Miescher. Currently it is a routine procedure in molecular biology or forensic analyses. ## Basic procedure of DNA extraction There are three basic and two optional steps in a DNA extraction: - Breaking the cells open, commonly referred to as cell disruption or cell lysis, to expose the DNA within. This is commonly achieved by chemical and physical methods - - Removing membrane lipids by adding a detergent or surfactants which also serves in cell lysis. - Removing proteins by adding a protease (optional but often done). - Removing RNA by adding an RNase (almost always done). - DNA purification from detergents, proteins, salts and reagents used during cell lysis step. The most commonly used procedures are: - Ethanol precipitation usually by ice-cold ethanol or isopropanol. Since DNA is insoluble in these alcohols, it will aggregate together, giving a pellet upon centrifugation. Precipitation of DNA is improved by increasing of ionic strength, usually by adding sodium acetate. - Phenol-chloroform extraction in which phenol denatures proteins in the sample. After centrifugation of the sample, denaturated proteins stay in organic phase while aqueous phase containing nucleic acid is mixed with the chloroform that removes phenol residues from solution. (Note: for DNA isolation in used phenol buffered to pH 8, RNA must be isolated using acidic phenol.) - Minicolumn purifications that relies on the fact that the nucleic acid may bind (adsorption) to the solid phase (silica or other) depending on the pH and the salt content of the buffer. ## Refinements of the technique include adding a chelating agent to sequester divalent cations such as, Mg²⁺ and Ca²⁺, which prevents enzymes like DNase from degrading the DNA. Cellular and histone proteins bound to the DNA can be removed either by adding a protease or by having precipitated the proteins with sodium or ammonium acetate, or extracted them with a phenol-chloroform mixture prior to the DNA-precipitation. After isolation, the DNA is dissolved in slightly alkaline buffer, usually in the TE buffer, or in ultra-pure water. ## Special Types of DNA Extractions Specific techniques must be chosen for isolation of DNA from some samples. ### Typical samples with complicated DNA isolation are: - archaeological samples containing partially degraded DNA, see ancient DNA - samples containing inhibitors of subsequent analysis procedures, most notably inhibitors of PCR, such as humic acid from soil, indigo and other fabric dyes or haemoglobin in blood - samples from microorganisms with thick cellular wall, for example yeast Extrachromosomal DNA is generally easy to isolate, especially plasmids may be easily isolated by cell lysis followed by precipitation of proteins, which traps chromosomal DNA in insoluble fraction and after centrifugation, plasmid DNA can be purified from soluble fraction. A Hirt DNA Extraction is an isolation of all extrachromosomal DNA in a mammalian cell. The Hirt extraction process gets rid of the high molecular weight nuclear DNA, leaving only low molecular weight mitochondrial DNA and any viral episomes present in the cell. ## Detecting DNA - A diphenylamine (DPA) indicator will confirm the presence of DNA. This procedure involves chemical hydrolysis of DNA: when heated (e.g. 295 °C) in acid the reaction requires a deoxyribose sugar and therefore is specific for DNA. Under these conditions, the 2-deoxyribose is converted to ω-hydroxylevulinyl aldehyde, which reacts with the compound, diphenylamine, to produce a blue-colored compound. DNA concentration can be determined measuring the intensity of absorbance of the solution at the 600 nm with a spectrophotometer and comparing to a standard curve of known DNA concentrations. - Measuring the intensity of absorbance of the DNA solution at wavelengths 260 nm and 280 nm is used as a measure of DNA purity. DNA absorbs UV light at 260 and 280 nanometres, and aromatic proteins absorb UV light at 280 nm; a pure sample of DNA has a ratio of 1.8 at 260/280 and is relatively free from protein contamination. A DNA preparation that is contaminated with protein will have a 260/280 ratio lower than 1.8. - DNA can be quantified by cutting the DNA with a restriction enzyme, running it on an agarose gel, staining with ethidium bromide or a different stain and comparing the intensity of the DNA with a DNA marker of known concentration. - Using the Southern blot technique, this quantified DNA can be isolated and examined further using PCR and RFLP analysis. These procedures allow differentiation of the repeated sequences within the genome. It is these techniques which forensic scientists use for comparison, identification, and analysis. ## Proteinase K: - Enzyme that digests proteins - Isolated from fungus - Stable across wide range of PH&Temperature - Activity is increased at elevated Temp.up to55° C - It is suited to short digestion time - EDTA does not interfere with its action - Proteins will be completely digested - Addition of proteinase K to nucleic acid preparations rapidly inactivates nucleases that might degradeDNA&RNA during purification ## 2-Mercaptoethanol: (also β-mercaptoethanol, BME, 2BME, 2-ME or β-met) - It is the chemical compound with the formula HOCH2CH2SH. ME or βME, as it is commonly abbreviated - It is used to reduce disulfide bonds - Can act as a biological antioxidant ### Denaturing ribonucleases 2-Mercaptoethanol is used in some RNA isolation procedures to: - Eliminate ribonuclease released during celllysis. - Disulfide bonds make ribonucleases very stable enzymes, so 2-mercaptoethanol is used to reduce these disulfide bonds and irreversibly denature the proteins. This prevents them from digesting the RNA during its extraction procedure. ## Terms - **2' deoxyribonucleoside triphosphate** - The building blocks of DNA replication. A five-membered, oxygen-containing ribose sugar ring that has three phosphate groups attached to its 5’ carbon and either an adenine, cytosine, guanine, or thymine base group attached to its 1’ carbon - **Daughter strand** - Refers to the newly synthesized strand of DNA that is copied via the addition of complementary nucleotides from one strand of pre-existing DNA during DNA replication - **DNA Helicase** - The enzyme responsible for separating the two strands of DNA in a helix so that they can be copied during DNA replication. - **DNA Ligase** - The enzyme responsible for sealing together breaks or nicks in a DNA strand. Responsible for patching together Okazaki fragments on the lagging strand during DNA replication. - **DNA Polymerase** - The enzyme responsible for catalyzing the addition of nucleotide substrates to DNA both during and after DNA replication. - **Primase** - The enzyme responsible for initiating synthesis of RNA primers on the lagging strand during DNA replication. - **Holoenzyme** - A term used to describe a collection of different enzymes that work together in a given process such as DNA replication. - **Lagging strand** - In DNA replication, the strand of pre-existing DNA that is oriented in the 5' to 3' direction with respect to the direction of replication on which synthesis is discontinuous. - **Leading strand** - In DNA replication, the strand of pre-existing DNA that is oriented in the 3' to 5' direction with respect to the direction of replication on which replication is continuous. - **Okazaki fragment** - Short stretches of newly synthesized DNA found on the lagging strand during DNA replication. - **Origin of replication** - Site of initiation of DNA replication. Short, usually internal stretch in a DNA helix that opens so that each strand is separate for DNA replication. - **Parent strand** - In DNA replication, refers to the pre-existing single strand of DNA that is copied into a new strand of DNA via complementary base pairing. - **Replication fork** - Term used to describe the junction at which nucleotide substrates are being added to a growing DNA chain during DNA replication. Its shape resembles a "Y" where the two branches represent single stranded daughter strands of DNA and the base represents helical DNA. - **RNA Primer** - Short stretches of ribonucleotides (RNA substrates) found on the lagging strand during DNA replication. Helps initiate lagging strand replication and are later removed.